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Methods and products for fusion protein synthesis

a technology of fusion protein and protein synthesis, which is applied in the field of methods and products for fusion protein synthesis, can solve the problems of limiting existing methods, errors in protein synthesis and misfolding, and numerous problems in clustering different kinds of proteins into precise artificial “fusion proteins” , to achieve the effect of simple and scalable method and high yield

Active Publication Date: 2020-01-07
OXFORD UNIV INNOVATION LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The present invention provides advantages because the peptide linkers used to join proteins together can be genetically encoded. This allows for complete control over the peptide linker sequence and reduces the risk of immunogenicity. The invention also provides nucleic acid molecules that encode the peptide linker or the polypeptides it connects together. These nucleic acid molecules can be introduced into cells for expression of the fusion protein or the individual polypeptides. Overall, the invention enhances the accuracy and safety of protein-based therapies.

Problems solved by technology

However, clustering different kinds of proteins into precise artificial “fusion proteins” has encountered numerous problems.
For instance, individual proteins or protein domains can be joined genetically into one long open reading frame, but errors in protein synthesis and misfolding soon become limiting.
Accordingly, existing methods are limited, insofar as they commonly result in undefined mixtures of fusion proteins that are difficult to separate and / or fusion proteins that are not stable across a variety of environments, e.g. in reducing conditions.
However, no existing fusion protein synthesis method has been able to fulfil these criteria.

Method used

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  • Methods and products for fusion protein synthesis
  • Methods and products for fusion protein synthesis
  • Methods and products for fusion protein synthesis

Examples

Experimental program
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Effect test

example 1

d Synthesis of Cognate Pairs of Peptide Linkers that Form Spontaneous Isopeptide Bonds

[0271]RrgA (SEQ ID NO: 21) is an adhesin from Streptococcus pneumoniae, a Gram-positive bacterium which can cause septicaemia, pneumonia and meningitis in humans. A spontaneous isopeptide bond forms in the D4 immunoglobulin-like domain of RrgA between residues Lys742 and Asn854 (FIG. 2). The inventor split the D4 domain into a pair of peptide linkers termed SnoopTag (residues 734-748, SEQ ID NO: 1) and a protein which we named SnoopCatcher (residues 749-860, SEQ ID NO: 2).

[0272]However, the inventors founds that it was necessary introduce two mutations in to the SnoopCatcher peptide linker in order to form a stable pair of peptide linkers for use in the invention. In this respect, the inventor introduced the G842T mutation in SnoopCatcher to stabilize a β-strand and the D848G to stabilize a hairpin turn close to the reaction site.

[0273]The SnoopTag peptide was expressed as a recombinant polypeptide...

example 2

ting the Cross-Reactivity of Pairs of Peptide Linkers

[0279]A peptide tag and binding partner, SpyTag and SpyCatcher (SEQ ID NOs: 13 and 14), that react spontaneously to form an isopeptide bond have been developed previously (WO2011 / 098772).

[0280]SnoopTag has a reactive Lys, whereas SpyTag has a reactive Asp, so the inventor hypothesized that the SnoopTag / SnoopCatcher and SpyTag / SpyCatcher pairs would be fully orthogonal, i.e. would not show cross-reactivity. Upon mixing the peptide linkers in various combinations, it was found that each cognate pair of peptide linkers reacted efficiently, but found no trace of cross-reaction between pairs was found, even after overnight incubation (FIG. 7). This result confirmed that the SnoopTag / SnoopCatcher pair is orthogonal to SpyTag / SpyCatcher.

[0281]The inventor also tested the PsCsTag / PsCsCatcher pair and RrgATag / RrgACatcher pair for cross-reactivity with the SnoopTag / SnoopCatcher and SpyTag / SpyCatcher pairs. As shown in FIG. 8, parts A and B,...

example 3

of Fusion Proteins Using Two Orthogonal Pairs of Peptide Linkers

[0282]The inventors used the “Spy” and “Snoop” pairs of peptide linkers to demonstrate that such orthogonal pairs of peptide linkers can be used successfully to synthesise fusion proteins.

[0283]The interaction of E. coli MBP with amylose resin is widely used in affinity purification: MBP-fusions typically fold and express well and show low non-specific resin binding. MBP shows selective mild elution using maltose, avoiding need for protease removal. The affinity of wild-type MBP for maltose is 1.2 μM, which is practical for protein purification but insufficient for multiple rounds of washing and chain extension in fusion protein synthesis. Therefore, the inventors developed a mutated MBP to improve its maltose-binding stability. Firstly, the inventors modified the polypeptide sequence by introducing mutations A312V and I317V and deleting residues 172, 173, 175 and 176). Secondly, the MBP mutant (SEQ ID NO: 70) was tande...

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Abstract

A method of producing a fusion protein is provided. The method includes: a) contacting a first protein with a second protein under conditions that enable the formation of an isopeptide bond between said proteins to form a linked protein; and b) contacting the linked protein from (a) with a third protein under conditions that enable the formation of an isopeptide bond between said third protein and said linked protein to form a fusion protein. Peptide linkers and the use of orthogonal pairs of said linkers in the synthesis of fusion proteins are also provided. Recombinant proteins comprising said linkers, nucleic acid molecules encoding said proteins and linkers, vectors comprising said nucleic acid molecules and host cells comprising said vectors and nucleic acid molecules are also contemplated.

Description

CROSS-REFERENCE[0001]This application is a section 371 U.S. National phase of PCT / GB2016 / 051640, filed Jun. 3, 2016 which claims priority from GB 1509782.7, filed Jun. 5, 2015, which is incorporated by reference in its entirety.FIELD OF INVENTION[0002]The present invention relates to the synthesis (i.e. production, generation or assembly) of a fusion protein (i.e. a polymer comprising two or more covalently linked proteins, as defined below) and in particular to the modular (e.g. step-wise) synthesis of a fusion protein using orthogonal pairs of peptide linkers which react to form isopeptide bonds. The invention concerns the provision of a new method for synthesizing fusion proteins, particularly solid-phase synthesis. The method can advantageously be used in the production of a variety of products comprising fusion proteins, e.g. fusion protein arrays. The present invention also provides peptide linkers and the use of orthogonal pairs of said linkers in the synthesis of fusion prot...

Claims

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Application Information

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Patent Type & Authority Patents(United States)
IPC IPC(8): C07K14/31C07K14/315C07K1/04
CPCC07K1/04C07K14/3156C07K14/315C40B40/10C12P21/00C07K2319/90
Inventor HOWARTH, MARK
Owner OXFORD UNIV INNOVATION LTD
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